Rapid measurement of macronutrients in breast milk: How reliable are infrared milk analyzers?

Abstract

Background & aims: Significant biological variation in macronutrient content of breast milk is an important barrier that needs to be overcome to meet nutritional needs of preterm infants. To analyze macronutrient content, commercial infrared milk analyzers have been proposed as efficient and practical tools in terms of efficiency and practicality. Since milk analyzers were originally developed for the dairy industry, they must be validated using a significant number of human milk samples that represent the broad range of variation in macronutrient content in preterm and term milk. Aim of this study was to validate two milk analyzers for breast milk analysis with reference methods and to determine an effective sample pretreatment. Current evidence for the influence of (i) aliquoting, (ii) storage time and (iii) temperature, and (iv) vessel wall adsorption on stability and availability of macronutrients in frozen breast milk is reviewed.

Methods: Breast milk samples (n = 1188) were collected from 63 mothers of preterm and term infants. Milk analyzers: (A) Near-infrared milk analyzer (Unity SpectraStar, USA) and (B) Mid-infrared milk analyzer (Miris, Sweden) were compared to reference methods, e.g. ether extraction, elemental analysis, and UPLC-MS/MS for fat, protein, and lactose, respectively.

Results: For fat analysis, (A) measured precisely but not accurately (y = 0.55x + 1.25, r(2) = 0.85), whereas (B) measured precisely and accurately (y = 0.93x + 0.18, r(2) = 0.86). For protein analysis, (A) was precise but not accurate (y = 0.55x + 0.54, r(2) = 0.67) while (B) was both precise and accurate (y = 0.78x + 0.05, r(2) = 0.73). For lactose analysis, both devices (A) and (B) showed two distinct concentration levels and measured therefore neither accurately nor precisely (y = 0.02x + 5.69, r(2) = 0.01 and y = -0.09x + 6.62, r(2) = 0.02 respectively). Macronutrient levels were unchanged in two independent samples of stored breast milk (-20 °C measured with IR; -80 °C measured with wet chemistry) over a period of 14 months.

Conclusions: Milk analyzers in the current configuration have the potential to be introduced in clinical routine to measure fat and protein content, but will need major adjustments.

Keywords: Fat; Freeze thaw cycle; Lactose; Protein; Stability; Validation study.

Fig. 1

Flow chart depicting the origin of the analyzed samples and corresponding chemical and IR measurements.

Fig. 2

Validation of IR methods for fat content: upper panel correlates fat content analyzed from reference method (ether extraction) with (A) Near-IR spectroscopy (n = 851) and (B) Mid-IR spectroscopy (n = 768) (dash line indicates line of identity); lower panel shows Bland–Altman plots indicating the difference between fat values obtained by the reference method (x-axis) and Near-IR spectroscopy or Mid-IR spectroscopy represented in y-axis.

Fig. 3

Validation of IR methods for protein content: upper panel correlates true protein content analyzed from reference method (elemental analysis) with (A) Near-IR spectroscopy (n = 812) and (B) Mid-IR spectroscopy (n = 754) (dash line indicates line of identity); lower panel shows Bland–Altman plots indicating the difference between protein values obtained by the reference method (x-axis) and Near-IR spectroscopy or Mid-IR spectroscopy represented in y-axis.

Fig. 4

Validation of IR methods for lactose content: upper panel correlates lactose content analyzed from the reference method (UPLC-MS/MS) with (A) Near-IR spectroscopy (n = 661) and (B) Mid-IR spectroscopy (n = 622) (dash line indicates line of identity); lower panel is Bland–Altman plots showing the difference between lactose values obtained by the reference method (x-axis) and Near-IR spectroscopy or Mid-IR spectroscopy represented in y-axis.

Fig. 5

Change in fat, protein, and lactose readings in breast milk samples (n = 3) over 90 min that have been homogenized at different settings (i.e. homogenized for 5, 10, 15, and 30 s, shaken by hand, and vortex for 1 min) using Near-IR spectroscopy. The average of the differences between each measurement and the initial (1 min) measurement (homogenized for 30 s) was calculated.

Fig. 6

Change in fat, protein, and lactose readings in breast milk (n = 10) over 10 min and 5 min analyzed by Near-IR and Mid-IR spectroscopy, respectively. Samples obtained from the same procedure of (A). (Measurement at 1 min mark is considered as immediate measurement due to the fact that it takes about 1 min to generate the data).

Fig. 7

(A) Imaging the change in fat droplet sizes of breast milk samples that correspond to different homogenization procedures; i.e. homogenized for. 5, 15, and 30 s, shaken by hand, and vortexed for 1 min, (B) Time-course analysis of change in fat droplet size (i.e. 0, 20, and 90 min) after homogenization for 5, 15, and 30 s.

Fig. 8

Correlation of the Mid-IR (Miris) and Near-IR (Unity) devices for fat (A), protein (B) and carbohydrates (C).

Fig. 9

Recalibration of the Mid-IR (Miris) and Near-IR (Unity) devices for fat (A), protein (B) and carbohydrates (C) using the linear regression approach.

Fig. 10

Stability of human milk samples (quality control, −20 °C storage temperature) over 400 days.

Fig. 11

Impact of storage time: correlation of Near-IR (uncorrected readings) and wet lab methods for fat, lactose. Data are grouped by the lag of time between Near-IR and wet lab methods as indirect measure of nutritional degradation.